Modified Kluyveromyces yeasts, their preparation and use

ABSTRACT

The present invention concerns yeasts of the genus Kluyveromyces having one or more genetic modifications of at least one gene coding for a protease, said gene reducing or modifying the proteolytic actively of said yeasts, as well as their use as a cellular host for the secretion of recombinant proteins.

The present invention relates to new genetically modified yeasts belonging to the genus Kluyveromyces and to their use to produce advantageously recombinant proteins.

The advances accomplished in the field of molecular biology have made it possible to modify microorganisms in order to make them produce proteins of interest and for example heterologous proteins (mammalian proteins, artificial proteins, chimetic proteins, and the like). In particular, numerous genetic studies have been performed on the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. More recently, genetic tools have been developed so as to use the yeast Kluyveromyces as host cell for the production of recombinant proteins. The discovery of the plasmid pKD1, derived from K.drosophilarum (EP 241 435), has made it possible to develop a particularly advantageous host vector system for the secretion of recombinant proteins (EP 361 991, EP 413 622).

However, the application of this system of production is still limited, in particular by the problems of the efficacy of gene expression in these recombinant microorganisms, by the problems of stability of the plasmids and also by the problems of degradation of the recombinant products by the cells in which they are synthesized. A proteolysis phenomenon can indeed manifest itself during transit of the protein of interest in the secretory pathway of the recombinant yeast, or by the existence of secreted proteases or proteases present in the culture medium following an undesirable cell lysis which occurs during fermentation.

The applicant has now shown that it is possible to improve the levels of production of the said recombinant proteins, that is to say in their integral form, in Kluyveromyces yeasts, by modifying at least one gene encoding a cellular protease, and especially a protease transiting through the secretory pathway. Surprisingly, the applicant has furthermore shown that such modifications are particularly advantageous since they make it possible to increase the levels of production of recombinant proteins, and this is all the more advantageous since the said modification is without apparent effect on the growth rate and the viability of the modified cells under industrial fermentation conditions. Still surprisingly, the applicant has also shown that the said modifications do not affect the stability of the transformant yeasts, which makes it possible to use the said yeasts in a particularly advantageous manner to produce recombinant proteins.

The subject of the present invention is therefore yeasts of the genus Kluyveromyces having one or more genetic modifications of at least one gene encoding a protease, modifying the proteolytic activity of the said yeasts. Preferably, the genetic modification(s) render the said gene partially or totally incapable of encoding the natural protease. In another preferred embodiment of the invention, the gene(s) thus genetically modified encode a non-functional protease, or a mutant having a modified proteolytic activity spectrum. In another preferred embodiment of the invention, the gene(s) encoding the said proteases are placed under the control of a regulated promoter.

The yeasts of the genus Kluyveromyces according to the invention comprise the yeasts as defined by van der Walt [in: The Yeasts (1987) N. J. W. Kregervan Rij (ed): Elsevier: p.224], and preferably the yeasts K.marxianus var.lactis (K.lactis), K.marxianus var. marxianus (K.fragilis), K.marxianus var. drosophilarum (K.drosophilarum), K.waltii, and the like.

Genetic modification should be understood to mean more particularly any suppression, substitution, deletion or addition of one or more bases in the gene(s) considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or alternatively by exposing the said yeasts to a treatment by means of mutagenic agents. As mutagenic agents, there my be mentioned for example physical agents such as energetic radiation (X, g, ultra violet rays and the like), or chemical agents capable of reacting with various functional groups of the bases of DNA, and for example alkylating agents [ethyl methanesulphonate(EMS), N-methyl-N′-nitro-nitrosoguanidine, N-nitroquinoline 1-oxide (NQO)], bialkylating agents, intercalating agents and the like. Deletion is understood to mean any suppression of the gene considered. It may relate in particular to a part of the region encoding the said proteases and/or of all or part of the transcriptional promoter region. The genetic modifications can also be obtained by gens disruption, for example according to the procedure initially described by Rothstein [Meth. Enzymol. 101 (1983) 202]. In this case, the entire coding sequence will be preferably disrupted so as to allow the replacement, by homologous recombination, of the wild-type genomic sequence by a non-functional or mutant sequence.

The said genetic modification(s) may be located in the gene encoding the said proteases, or outside the region encoding the said proteases, for example in the regions responsible for the transcriptional expression and/or regulation of the said genes. The inability of the said genes to encode the natural proteases can manifest itself either by the production of a protein which is inactive because of structural or conformational modifications, or by the absence of production, or by thy production of a protease having a modified enzymatic activity, or alternatively by the production of the natural protease at an attenuated level or according to a desired mode of regulation.

Moreover, certain modifications such as point mutations are by nature capable of being corrected or attenuated by cellular mechanisms, for example during the replication of DNA preceding cell division. Such genetic modifications are thereby of limited interest at the industrial level since the phenotypic properties resulting therefrom are not perfectly stable. The applicant has now developed a process which makes it possible to prepare Kluyveromyces yeasts having one or more genetic modifications of at least one gene encoding a protease, the said modification(s) being segregationally stable and/or non-reversible. The yeasts having such modifications are particularly advantageous as cellular host for the production of recombinant proteins. The invention also makes it possible to produce yeasts in which the modification(s) made render the gene(s) considered totally or only partially incapable of producing a functional protease.

Preferably the yeasts according to the invention have one or more segregationally stable genetic modifications. Still according to a preferred embodiment, the genetic modification(s) its non-reversible. Still according to a preferred embodiment of the invention, the genetic modification(s) leave(s) no residual activity for the gens considered.

Preferably, the gene(s) encoding one or more proteases are chosen from the genes encoding proteases transiting through the secretorypathway of Kluyveromyces. Such proteases may be located in the endoplasmic reticulum, the compartment of the Golgi apparatus, the post-Golgi compartment, and for example the cellular vacuoles, the vesicles of the endosome, the secretion vesicles, or the extracellular medium.

As example of such genes, there may be mentioned the Kluyveromyces genes encoding a protease chosen from the families comprising protease A, protease B, or a carboxypeptidase (and for example carboxypeptidase Y or carboxypeptidase S), or alternatively endopeptidase KEX1 of K. lactis or a protease with similar activity, and for example protease YAP3 [Egel-Mitani et al., Yeast 6 (1990) 127], or more generally any other protease involved in the maturation of certain secreted proteins.

In a preferred embodiment of the invention, the considered gene(s) encode proteases which are not involved in the cleavage of the signal peptide of the recombinant proteins expressed in the form of preproteins. There may be mentioned by way of examples of particularly useful genes the genes for protease A, for protease B, and for carboxypeptidase Y of Kluyveromyces, whose cloning is described in the examples.

In another embodiment of the invention, the said protease(s) possess(es) a signal peptidase activity and the said genetic modification(s) allow their overexpression, which is particularly advantageous in the case or this step is a limiting step of the secretorypathway.

The subject of the invention is also any Kluyveromyces yeast as defined above into which an exogenous DNA sequence comprising one or more genes encoding a protein of interest which it is desired to express and/or secrete in the said yeast, has been introduced.

For the purposes of the present invention exogenous DNA sequence is understood to mean any DNA sequence introduced artificially into the yeast and encoding one or more proteins of interest. In particular, this may be complementary DNA (cDNA) sequences, artificial or hybrid sequences, or alternatively synthetic or semi-synthetic sequences, which are included in an expression cassette permitting synthesis in the said yeasts of the said protein(s) of interest. For example, this exogenous DNA sequence may include a region for initiation of transcription, regulated or otherwise in Kluyveromyces, so as to direct, when desirable, the expression of the said proteins of interest.

Preferably, the exogenous DNA sequence is included in a vector, which may be capable of autonomous replication in the yeast considered, or of the integrative type. More particularly, autonomously replicating vectors can be prepared from autonomously replicating sequences in Kluyveromyces, and for example this may be the plasmid pKD1 [Falcone et al., Plasmids 15 (1986) 248; Chen et al., Nucl. Acids Res. 14 (1986) 4471] characterized by a high segregational stability and especially in the various varieties of K. marxianus, or the plasmid pEW1 isolated in K. waltii [Chen et al., J. General Microbiol. 138 (1992) 337]. Autonomously replicating vectors can also be prepared from chromosomal sequences (ARS). As regards the integrarive-type vectors, these can be prepared from chromosomal sequences homologous to the said host yeast, so as to flank the genetic sequence encoding the said proteins of interest, and a genetic selectable marker, so as to orient the integration of the whole by homologous recombination. In a specific embodiment, the said homologous sequences correspond to genetic sequences derived from the coding region of the said protease, which makes it possible to replace by homologous recombination the original sequence of the said protease by the selectable marker and the exogenous DNA sequence, while permitting gene disruption of the said protease. In another embodiment, the expression cassette is integrated at the locus encoding the ribosomal RNAs (rDNA) permitting gens amplification of the said expression cassette [Bergkamp et al., Curr. Genet. 21 (1992) 365]. Still in another embodiment, the exogenous DNA sequence is integrated into the chromosome of the said host yeasts by non-homologous recombination.

The exogenous DNA sequence can be introduced into the yeast by the techniques practised by persons skilled in the art, and for example recombinant DNA techniques, genetic crossings, protoplast fusion, and the like. in a specific embodiment, the exogenous DNA sequence is introduced into Kluyveromyces yeasts by transformation, electroporation, conjugation, or any other technique described in the literature. As regards transformation of the Kluyveromyces yeasts, the technique described by Ito et al. [J. Bacteriol. 153 (1983) 163] can be used. The transformation technique described by Durrens et al. [Curr. Genet. 18 (1990) 7] using ethylene glycol and dimethyl sulphoxide is also effective. It is also possible to transform the yeasts by electroporation, according to the method described by Karube et al. [FEBS Letters 182 (1985) 90]. An alternative procedure is also described in Patent Application EP 361 991.

The said Kluyveromyces yeasts modified for their protease content by the techniques described above are advantageously used as host cells to produce recombinant proteins, and for example heterologous proteins of pharmaceutical or dietary interest. The said host yeasts are particularly advantageous since they make it possible to increase the quality and quantity of recombinant proteins which it is desired to produce and/or secrete, and since the said genetic modifications of the said cells do not affect the genetic and mitotic stability of the vectors for expression of the said recombinant proteins. Another subject of the invention therefore lies in a process for producing recombinant proteins according to which a yeast as defined above is cultured under conditions for expressing the protein(s) encoded by the exogenous DNA sequence, and the protein(s) of interest is (are) recovered. In a preferred embodiment, the said proteins of interest are secreted into the culture medium. As example, there may be mentioned naturally occurring proteins, or artificial proteins, and for example hybrid proteins. In this case, the use of yeast cells having a modified protease content is particularly advantageous because of the exposure of the hinge region between the various protein domains of the chimera. In a specific embodiment, the said artificial protein contains a peptide fused to one of the ends of the chimera and is particularly sensitive, for example during transit in the secretory pathway, to a proteolytic degradation by an N- or C-terminal exoprotease, and for example a carboxypeptidase. It is understood that the proteolytic degradation of the protein of interest can also result from any cellular protease, and for example cytoplasmic protease, released into the external medium because of an undesirable cell lysis during the fermentation process of the said recombinant yeasts. Genetic modification of the nucleotide sequence encoding such proteases can therefore also result in a particularly advantageous process for producing the said proteins of interest and is also claimed.

Preferably, the process according to the invention allows the production of proteins of pharmaceutical or dietary interest. As example, there may be mentioned enzymes (such as in particular superoxide dismutase, catalase, amylases, lipases, amidases, chymosin and the like, or any fragment or derivative thereof), blood derivatives (such as serum albumin, alpha- or beta-globin, coagulation factors, and for example factor VIII, factor IX, von Willebrand's factor, fibronectin, alpha-1 antitrypsin, and the like, or any fragment or derivative thereof), insulin and its variants, lymphokines [such as interleukins, interferons, colony-stimulating factors (G-CSF, GM-CSF, M-CSF and the like), TNF and the like, or any fragment or derivative thereof], growth factors (such as growth hormone, erythropoietin, FGF, EGF, PDGF, TGF, and the like, or any fragment or derivative thereof), apolipoproteins and their molecular variants, antigenic polypeptides for the production of vaccines (hepatitis, cytomegalovirus, Epstein-Barr virus, herpes virus and the like), or alternatively polypeptide fusions such as especially fusions containing a biologically active part fused to a stabilizing part. The proteins may comprise an exporting sequence for secretion.

Another subject of the present invention lies in a Kluyveromyces DNA fragment encoding a protease. The Applicant has indeed detected, isolated and characterized certain Kluyveromyces proteases and especially proteases transiting through the secretcry pathway. More preferably, one of the subjects of the invention relates to a Kluyveromyces protease chosen especially from proteases A, B, carboxypeptidase Y, as well as the family of seine proteases of the subtilisin type and of which one representative is the K. lactis KEX1 protease [Wésolowski-Louvel et al., Yeast A (1988) 71]. By way of example, the nucleotide sequences of the K. lactis genes encoding proteases A, B and carboxypeptidase Y were determined by the Applicant and are presented SEQ ID No. 5, 1 and 2 respectively. A restriction map of a chromosomal fragment encoding K. lactis protease A is also presented in FIG. 10. It is understood that any genetic variant of these protease genes, and their advantageous use for producing proteins of interest, also form part of the invention. The said variations may be of natural origin, but may also be obtained in situ or in vitro by genetic engineering techniques, or after treating the cells with a mutagenic agent, and include in particular point or multiple mutations, deletions, additions, insertions, hybrid proteases and the like. In a still more specific embodiment, the genetic variations may also relate to the regions for controlling the expression of the said proteases, for example so as to modify their levels of expression or their mode of regulation.

The subject of the invention is also any protein resulting from the expression of an exogenous DNA fragment as defined above.

The subject of the invention is also a process for preparing a genetically modified Kluyveromyces yeast and its advantageous use for producing proteins of interest. Preferably, the process of the invention consists in replacing the chromosomal gene(s) considered by a version modified in vitro.

The present invention will be more fully described with the aid of the following examples which should be considered as illustrative and non-limitative.

BRIEF DESCRIPTION OF THE DRAWING

The representations of the plasmids indicated in the following figures are drawn to a rough scale and only the restriction sites which are important for understanding the clonings carried out are indicated.

FIG. 1: Restriction map of the genomic insert of the plasmid pFP8. The position of the cleavage sites of the following endonucleases is indicated B=BamHI; G=BglIII; C=ClaI; E=EcoRI; H=HindIII; K=KpnI; N=NcoI; P=PstI; Pv=PvuII; T=SstI; X=XhoI. The fragment obtained after PCR amplification is represented as well as the mRNA of the S. cerevisiae PRB1 gene.

FIGS. 2A and B: Hybridization of the S. cerevisiae PRB1 probe with restriction fragments of the K. lactis genomic DNA. Top panelPanel A: photo of the agarose gel (1%) stained with ethidiumbromide and before transfer onto nylon filter, bottom panelPanel B: schematic representation of the signals obtained after hybridization of the filter with the radioactive probe. The numbering corresponds to the following digestions: 1=EcoRI; 2=BglII; 3=BglII+EcoRI; 4=HindIII; 5=HindIII+EcoRI; 6=PstI; 7=PstI+EcoRI; 8=SalI; 9=SalI+EcoRI; 10=BamHI; 11=BamHI+EcoRI.

FIGS. 3A and B: Hybridization of the S. cerevisiae PRB 1 probe with the 34 minipreparations of DNA (mixture of 10 clones each) after double restriction with the enzymes EcoRI and BglIII. Panel A: photo of the agarose gel (1%) stained with ethidiumbromide and before transfer onto nylon filter; Panel B: autoradiography of the nylon filter after hybridization with the S. cerevisiae PRB1 probe; subfractions “d”, “e” and “f” corresponding to an aliquot of the K. lactis genomic DNA fragments after total digestion with EcoRI+BglII and size fractionation by electroelution are indicated.

FIGS. 4A and B: Control hybridization of the positive clones 18-3 and 31-L Panel A: photo after running on 1% agarose gel of EcoRI+BglII digests of the DNA of clones 31-I (well no. 1), 18-3 (well no. 2) and 31-K (negative control, well no. 3). Panel B: autoradiography of the nylon filter corresponding to the preceding gel after hybridization with the S. cerevisiae PRB1 probe. P corresponds to the location of the undigested plasmid (pYG1224); the position of the BglII-EcoRI restriction fragment of about 0.7 kb hybridizing with the radioactive probe is indicated.

FIG. 5: Comparison of the protein sequence of the BglII-EcoRI fragment of K. lactis genomic DNA (residues Arg³⁰⁸ to Phe⁵³¹ of the peptide sequence SEQ ID No. 1) with the corresponding part of the S. cerevisiae PRB1 gene (SEQ ID NO. 8) (residues Arg¹⁰⁵ to Leu328). The asterisks indicate the amino acids conserved between the two sequences.

FIGS. 6A-D: Restriction maps of the genomic inserts of plasmids pYG1224, pYG1226 and pYG1227 (panel A); pYG1231 (panel B); pYG1237, pYG1238, pYG1239, pYG1240, pYG1241 and pYG1242 (panel C). The position of the cleavage sites of the following endonucleases is indicated: G=BglII; C=ClaI; S=SalI; E=EcoRI; H=HindIII; K=KpnI; P=PstI; V=EcoRV. Panel D: location of the coding phase of the K. lactis PRB1 gene; the vertical arrow indicates the rough position of the N-terminal end of the mature protein. The position of the codon presumed to be for initiation of translation and the position of the translational stop codon are indicated by an asterisk.

FIG. 7: Restriction map of the insert of the plasmid pC34. The box corresponds to the K. lactis genomic insert and the line corresponds to the sequences of the vector KEp6. The arrow indicates the position of the K. lactis PRC1 gene. The detailed part between the EcoRI and SalI sites corresponds to the sequenced region presented in SEQ ID NO 3. List of abbreviations: C=ClaI; H=HindIII; B=BamHI; E=EcoRI; P=PstI; S=SalI; Sp=SphI; Sau=Sau3A.

FIG. 8: Restriction map of the insert of the plasmid pA25/1. The box corresponds to the K. lactis genomic insert and the line corresponds to the sequences of the vector KEp6. The arrow indicates the rough position of the PRA1 gene as indicated by a Southern blot hybridization by means of specific 3′ and 5′ probes. List of abbreviations: C=ClaI; H=HindIII; B=BamHI; Sau=Sau3A; S=SalI; P=PstI; G=BglII; Xb=XbaI; Sn=SnaBI; E=EcoRI.

FIGS. 9A and B: Panel A: Restriction mat of the HindIII-EcoRI restriction fragment of the plasmid pYG1232. The position of the cleavage sites of the following endonucleases is indicated: G=BglII; S=SalI; E=EcoRI; H=HindIII; K=KpnI; X=XhoI; the HindIII cloning site is derived from the vector and is underlined. Panel B: Disruption of the K. lactis PRB1 gene by the selectable marker URA3 from S. cerevisiae. This Southern blot corresponds to the genomic DNA of K. lactis CBS 294.91 (uraA) after transformation by the BglII-EcoRI fragment of panel A and selection in the absence of uracil. Wells 1 to 3: genomic DNA of three transformants after BglII+EcoRI double restriction; the strain of well 3 is K. lactis Y750; well 4: genomic DNA of the strain CBS 294.91 after BglII+EcoRI double restriction. The radioactive probe used corresponds to the BglII-EcoRI fragment of the plasmid pYG1224 (FIG. 6A).

FIGS. 10A-D: Restriction map of the inserts of the plasmids pC34 [vector KEp6; panel a)], pYG154 [vector pIC-20R; panel b)] and pYG155 [vector pIC-20R; panel c)]. Panel d): fragment used for the disruption. The restriction sites in brackets were destroyed with the Klenow enzyme or with T4 DNA polymerase as indicated in the text. The box corresponds to the K. lactis genomic insert and the line corresponds to the sequences of the vectors. List of abbreviations: C=ClaI; H=HindIII; B=BamHI; E=EcoRI; P=PstI; S=SalI, Sp=SphI; Sau=Sau3A; Sm=SmaI; N=NcoI.

FIG. 11: Restriction map of the plasmid pYG105 and strategy for constructing the plasmid pYG1212. Abbreviations used: P, K. lactis LAC4 promoter T, transcriptional terminator; IR, inverted repeat sequences of the plasmid pKD1; LP, prepro region of HSA: Ap^(r) and Km^(r) designate respectively the genes for resistance to ampicillin (E. coli) and to G418 (Kluyveromyces).

FIG. 12: Comparison of the capacities of secretion of a truncated variant of human albumin in the K. lactis CBS 293.91 strains (wells 2, 4, 6, 8 and 10) or its disrupted mutant for the gene for protease B (strain Y750; wells 1, 3, 5, 7 and 9), after transformation with the plasmid pYG1212. The transformant cells are cultured in Erlenmeyer flasks in the presence of G418 (200 mg/l) for 2 days (wells 1, 2, 7 and 8), 4 days (wells 3, 4, 9 and 10), or 7 days (wells 5 and 6); wells 1 to 6 correspond to growth in YPD medium, and wells 7 to 10 correspond to growth in YPL medium. The spots are equivalent to 50 ml of culture supernatents.

GENERAL CLONING TECHNIQUES

The methods conventionally used in molecular biology, such as the preparative extractions of plasmid DNA, the centrifugation of plasmid DNA in caesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, extractions of proteins with phenol or phenol-chloroform, DNA precipitation in saline medium with ethanol or isopropanol, transformation in Escherichia coli, and the like are well known to persons skilled in the art and are widely described in the literature [Maniatis T. et al., “Molecular Cloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F. M. et al., (eds), “Current Protocols in Molecular Biology”, John Wiley & Sons, New York, 1987].

The restriction enzymes were provided by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used according to the recommendations of the suppliers.

The pBR322 and pUC type plasmids and the phages of the M13 series are of commercial origin (Bethesda Research Laboratories). The pIC type plasmids have been described by Marsh et al. [Gene 32. (1984) 481].

For the ligations, the DNA fragments are separated according to their size by electrophoresis on agarose or acrylamide gels, extracted with phenol or with a phenol/chloroformmixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the supplier.

The filling of the protruding 5′ ends is carried out by the Klenow fragment of DNA polymerase I of E. coli (Biolabs) according to the specifications of the supplier. The destruction of the protruding 3′ ends is carried out in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer. The destruction of the protruding 5′ ends is carried out by a controlled treatment with S1 nuclease. The exonuclease Ba131 is used according to the recommendations of the supplier (Biolabs).

The oligodeoxynucleotides are synthesized chemically according to the phosphoramidite method using β-cyanoethyl protective groups [Sinha et al., Nucleic Acids Res. 12 (1984) 4539]. After synthesis, the protective groups are removed by treatment with ammonium hydroxide and two precipitations with butanol make it possible to purify and concentrate the oligodeoxynucleotides [Sawadogo and Van Dyke, Nucleic Acids Res. 19 (1991) 674]. The DNA concentration is determined by measuring the optical density at 260 nm.

Site-directed mutagenesis in vitro with synthetic oligodeoxynucleotides is carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749] using the kit distributed by Amersham.

The DNA fragment used to serve as molecular probe on the K. lactis genomic DNA is amplified in vitro by the PCR technique [Polymerase-catalysed Chain Reaction, Saiki R. K. et al., Science 230 (1985) 1350; Mullis K. B. and Faloona F. A., Meth. Enzym. 155 (1987) 335] on the S. cerevisiae DNA. The amplification is automated (40 amplification cycles) and is carried out in a Perkin Elmer Cetus apparatus (DNA thermal cycler) Using the Taq polymerase (isolated from the archaebacterium Thermophilus aquaticus) provided by the company Perkin Elmer. Each amplification cycle comprises three stages:

1) A stage for denaturation of DNA at 910° C.;

2) A stage for hybridization of oligodeoxynucleotide primers onto the template DNA. The hybridization temperature is chosen five to ten degrees below the melting temperature of the oligodeoxynucleotides (T_(1/2)). For oligodeoxynucleotides of about 20 mer in size, T_(1/2)=2x(A+T)+4x (C+G) [Itakura et al., Ann. Rev. Blochem. 53 (1984) 323].

3) A stage for synthesis of complementary DNA by Taq polymerase at 72° C.

The preparation of the radioactive nucleotide probes is carried out by incorporation of radioactive adCTP (phosphorus 32) along the length of the molecule neosynthesized from 20 ng of DNA using the “Random Primed DNA Labeling” kit marketed by the firm Boehringer.

The transfers of DNA onto nylon membrane (Biodyne, Pall. St Germain en Laye) or nitrocellulose (Schleicher & Schuell, Dassel) are carried out according to the method initially developed by Southern [J. Mol. Biol. 98 (1979) 503]. The hybridization and washing conditions used depend on the nature of the probe used: under heterologous conditions (K. lactis genomic DNA hybridized with a probe from S. cerevisiae for example), the hybridization and the washes are carried out under conditions which are not very stringent (hybridization for 15 hours at 40° C. without formamide in 5X SSC/5X Denhart, the filter is then washed 3 times in 5X SSC/1% SDS at 40° C. for 15 minutes, then once in 0.25X SSC/1% SDS for 10 minutes); under homologous conditions (K. lactis genomic DNA hybridized with a probe from K. lactis for example), the hybridization and the washes are carried out under more stringent conditions (hybridization for 15 hours at 40° C. in 5X SSC/SX Denhart/50% formamide, the filter is then washed 3 times in 5X SSC/1% SDS at 40° C. for 15 minutes, then once in 0.2X SSC/1% SDS for 10 minutes).

The verification of the nucleotide sequences is carried out on plasmid DNA with the “Sequenase version 2.0” kit from the Company United States Biochemical Corporation, according to the method by Tabor and Richardson [Proc. Natl. Acad. Sci. USA 84 (1987) 4767]. This technique is a modification of the method initially described by Sanger et al. [Proc. Natl. Acad. Sci. USA 74 (1977) 5463].

The transformations of K. lactis with DNA from the plasmids for expression of the proteins of the present invention are carried out by any technique known to persons skilled in the art, and of which an example is given in the text.

Unless otherwise stated, the bacteria strains used are E. coli MC 1060 (lacIPOZYA, X74, galU, galK, strA^(r)), E. coli TG1 (lac, proA, B, supE, thi,hsdDS/FtraD36, proA⁺B⁺, lacI¹,lacZ, M15), or E. coli JM101 [Messing et al., Nucl. Acids Res. 9 (1981) 309].

The yeast strains used belong to the budding yeasts and more particularly to yeasts of the genus Kluyveromyces. The K. lactis MW98-8C (a, uraA, arq, lys, K⁺, pKD1°), K. lactis CBS 293.91, K. lactis CBS 294.91 (uraA), and K. lactis CBS 2359/152 [a, metA, (k1, k2); Wésolowski et al., Yeast 4 (1988) 71] strains were particularly used; a sample of the MW98-8C strain was deposited on 16 Sep. 1988 at Centraalbureau voor Schimmelkulturen (CBS) at Baarn (The Netherlands) where it was registered under the number CBS579.88.

The preparation of yeast genomic DNA is essentially derived from the technique by Hoffman and Winston [Gene 57 (1987) 267] and is described in detail in the text.

The yeast strains transformed with the expression plasmids encoding the proteins of the present invention are cultured in Erlenmeyer flasks or in 2 l pilot fermenters (SETRIC, France) at 28° C. in rich medium (YPD: 1% yeast extract, 2% Bactopeptone, 2% glucose; or YPL: 1% yeast extract, 2% Bactopeptone, 2% lactose) with constant stirring.

EXAMPLES Example 1 CLONING OF THE K. LACTIS PROTEASE B GENE

E.1.1. Production of a probe by enzymatic amplification in vitro of a DNA sequence.

An enzymatic amplification by PCR is carried out starting with the plasmid pFP8 [Moehle etal., Genetics 115 (1987) 255; FIG. 1], E. coli/S. cerevisiae shuttle vector derived from YEp13 and carrying the S. cerevisiae PRB1 gens and the oligodeoxynucleotides 5′-TGACACTCAAAATAGCG-3′ (SEQ ID NO. 9) the codon corresponding to the Asp³ residue is underlined) and 5′-AATATCTCTCACTTGAT-3′ (SEQ ID NO. 10) (the codon of the complementary strand corresponding to the residue Ile³⁴⁸ is underlined). The temperature for hybridization of the oligodeoxynucleotides is 45° C. and the reaction volume of 100 ml comprises: 10 ng of plasmid pFPS, the oligodeoxynucleotide primers, 10 ml of 10X PCR buffer [Tris-HCl pH=8.5 (100 mM); MgC12 (20 mM); KCl (100 EM); gelatin (0.01%)), 10 ml dNTP (dATP+dCTP+dGTP+dTTP, each at a concentration of 10 mM)], and 2.5 units of Taq polymerase. The addition of a drop of paraffin oil makes it possible to avoid evaporation during elevations of temperature during the amplification cycles. A DNA fragment of 1.039 base pairs is obtained, whose identity is verified by analysis of the positions of certain restriction sites and corresponding to virtually the entire amino acid sequence of the mature form of protease B (Asp³ to Ile³⁴⁸). This fragment is then purified by electroelution after migration on a 0.8% agarose gel and is used to prepare the radioactive probe according to the “Random Priming” method.

E.1.2. Preparation of yeast genomic DNA.

The yeasts (strain K. lactis MW98-SC) in stationary growth phase are centrifuged. After washing the pellet in sterile water, the latter is taken up in a solution containing: 2% Triton X100 (v/v); 1% SDS (w/v); 100 mMNaCl; 10 mMTris-HCl (pH=8); 1 mM EDTA. The yeasts are then ground in the presence of phenolchloroform by the mechanical action of glass beads added to the mixture which is vortexed for 2 minutes. The aqueous phase is then recovered after centrifugation and precipitated by addition of 2.5 volumes of ethanol. The DNA is taken up in TE so as to be subjected to purification on a caesium gradient. Starting with 1 litre of culture, 1 mg of high molecular weight genomic DNA (>>20kb) is obtained.

E.1.3. Search for the gene byhybridizationunder low stringency conditions.

The genomic DNA preparation is subjected to total digestion with restriction enzymes whose sites are present in the multiple cloning site of the vector pIC-20H. A preliminary result shows that only conditions which are not very stringent (hybridization for 15 hours at 40° C. without formamide in 5X SSC/SX Denhart, then 3 washes in 5X SSC/1% SDS at 40° C. for 15 minutes, then 1 wash in 0.25X SSC/1% SDS for 10 minutes) make it possible to visualize an EcoRI fragment of 1.8 kb hybridizing with the S. cerevisiae PRB1 probe. A second Southern blotting is carried out in which each well contains 12 mg of genomic DNA cleaved for 15 hours with 20 units of EcoRI and 20 units of a second restriction enzyme (FIG. 2). Under the hybridization and washing conditions previously defined, a genomic fragment of about 700 bp and derived from the EcoRI+BglII double digestion hybridizes with the S. cerevisiae PRB1 probe (FIG. 2, well no. 3). Likewise, a BglII restriction fragment of greater size (about 1.3 kb) hybridizes with this probe (FIG. 2, well no. 2). The fragment of about 700 bp detected after digestion of the genomic DNA with EcoRI+BglII is therefore a BglII-EcoRI restriction fragment. The other restrictions appear to be less important since they generate either fragments which are smaller in size than that obtained by the EcoRI+BglII double digestion, or a fragment of identical size (1.8 kb) to that generated after digestion with EcoRI alone (FIG. 2, well no. 1).

The cloning of this BglII-EcoRI asymmetric fragment of 700 bp makes it possible to obtain a fraction of the K. lactis PRB1 gene which can then serve as homologous probe for cloning the missing part(s) of the gene. To clone this fragment, 100 mg of genomic DNA are treated for 15 hours with 100 units of EcoRI and BglII endonucleases, then run on preparative agarose gel (1%). The fraction of the gel comprising the fragments whose size is between 500 and 1,000 bp is then cut into three subfractions (subfraction {fraction (500/700)} bp; subfraction “e”: {fraction (700/800)} bp; subfraction “d”: {fraction (800/1000)} bp; FIG. 3). A Southern blotting carried out after running an aliquot of these subfractions and hybridization with the S. cerevisiae PRB1 probe shows a hybridization signal of the expected size (700 bp) and particularly intense with the subfraction “e” ({fraction (700/800)} bp). A genomic library restricted to the BglII-EcoRI fragments of this subfraction (mini-genomic library) is therefore constructed by cloning the BglII-EcoRI restriction fragments into the corresponding sites of the vector pIC-20H. The transformation of the ligation in E. coli gives 90% of white clones in LB dishes supplemented with ampicillin and X-gal. 340 clones (including about 30 blue clones) are then subcultured on the same medium so as to isolate them. The 340 clones of the restricted library are then divided into 34 mixtures each corresponding to 10 different clones and their DNA is digested with EcoRI+BglII, run on agarose gel, transferred onto membrane so as to then be hybridized with the S. cerevisiae PRB1 probe under hybridization and washing conditions which are not very stringent. FIG. 3 shows this Southern blot where two mixtures (no. 18 and no. 31) show a hybridization signal of the expected size (about 700 bp). The same operation is carried out in order to analyse separately the 10 clones of mixtures no. 18 and no. 31: clone no. 3 is the only clone of the mixture no. 18 to have a hybridization signal at the expected size (clone 18-3);. in a similar manner, only clone I of the mixture 31 (clone 31-I) gives a hybridization signal at the expected size. A last Southern blotting is carried out starting with the DNA of clones 18-3 and 31-I (positive signal) and a negative clone (clone K of mixture no. 31). Under the hybridization and washing conditions previously defined, only clones 18-3 and 31-I confirm the presence of a positive signal after EcoRI+BglII double digestion, whose size seems to be strictly equivalent (FIG. 4).

E.1.4. Identification of the gene.

The nucleotide sequence of the BglII-EcoRI fragment of clone 18-3 is produced in order to demonstrate that this fragment indeed corresponds to a fraction of the K. lactis PRB1 gene.

A rough restriction map of the plasmid pYG1224 from clone 18-3 is first produced and reveals the presence of an apparently unique SalI site at the centre of the BglII-EcoRI fragment. The BglII-SalI (about 350 bp) and SalI-EcoRI (about 300 bp) fragments of the plasmid pYG1224 are then cloned into the vector pUC19, which generates the plasmids pYG1226 and pYG1227 respectively (FIG. 6, panel A). The inserts of these plasmids are then sequenced in full using “universal primers”. As indicated in FIG. 5, the BglII-EcoRI fragment of the plasmid pYG1224 contains an open reading frame (225 residues) which exhibits sequence homologies with a fragment of the S. cerevisiae PRB1 gene (Arg¹⁰⁵ to Leu³²⁸). The presence of such a homology, as well as the strict conservation of the amino acids invariably found in the serine proteases of the subtilisin family demonstrate that the genomic DNA fragment carried by the plasmid pYG1224 indeed corresponds to a fragment of the K. lactis PRB1 gene.

E1.5. Cloning of the 3′ part of the gene.

The BglII-EcoRI fragment of the plasmid pYG1224 contains a unique KpnI restriction site located downstream of the BglII site (FIG. 6, panel A). The KpnI-EcoRI restriction subfragment of about 665 nucleotides is therefore generated from this fragment, isolated by electroelution after running on a 1% agarose gel and radioactively labelled by the “random priming” method. This radioactive probe is then used to determine the size of the restriction fragments of the K. lactis genomic DNA which include it. A KpnI-BglII fragment of about 1.2 kb is thus detected after hybridization and washing under stringent conditions (hybridization for 15 hours at 40° C. in 5X SSC/5X Denhart/50% formamide, then 3 washes in 5X SSC/1% SDS at 40° C. for 15 minutes, then 1 wash in 0.2X SSC/1% SDS for 10 minutes). A restricted library of K. lactis genomic DNA (KpnI-BglII restriction fragments of between 1 and 1.5 kb in size) is then constructed according to Example E1.3. and the restriction fragment hybridizing with the probe is cloned between the KpnI and BamHI sites of the vector pIC-20H, which generates the plasmid pYG1231 (FIG. 6, panel B). The genomic insert of this plasmid is then sequenced using the oligodeoxynucleotide Sq2101 (5′-GACCTATGGGGTAAGGATTAC-3′) (SEQ ID NO. 11) as primer. This oligodeoxynucleotide corresponds to a nucleotide sequence present in the BglII-EcoRI fragment of the plasmid pYG1224 and located at about 30 nucleotides from the EcoRI site. It therefore makes it possible to determine the nucleotide sequence situated in 3′ of this restriction site, and especially the sequence located between the EcoRI site and the translational stop codon of the messenger RNA corresponding to the K. lactis PRB1 gene.

E.1.6. Cloning of the 5′ part of the gene.

The nucleotide sequence produced in E.1.5. demonstrates the existence of a HindIII restriction site located between the EcoRI site and the translational stop codon. The use of the KpnI-EcoRI restriction fragment corresponding to the C-terminal part of the K. lactis PRB1 gene as radioactive probe on the K. lactis genomic DNA digested with HindIII and a second enzyme makes it possible to identify, by Southern blotting, a HindIII-EcoRV fragment of about 1.7 kb which hybridizes with this probe. This restriction fragment is first cloned between the EcoRV and HindIII sites of the vector pIC-20R, thereby generating the plasmid pYG1237. A restriction map of the genomic DNA insert contained in the plasmid pYG1237 is produced (FIG. 6, panel C), and the following plasmids are generated: pYG1238 (plasmid pYG1237 deleted in relation to its PstI fragment), pYG1239 (PstI fragment of pYG1237 in the vector pUC19), pYG1240 (plasmid pYG1237 deleted in relation to its KpnI fragment), pYG1241 (plasmid pYG1237 deleted in relation to its ClaI fragment) and pYG1242 (plasmid pYG1237 deleted in relation to its SalI fragment; FIG. 6, panel C). The genomic inserts of these various plasmids are then sequenced with the aid of universal primers and the oligodeoxynucleotide Sq2148 (5′-GCTCGGCAACATATTCG-3′) (SEQ ID NO. 12) which makes it possible to sequence the region situated immediately in 5′ of the BglII site. This strategy makes it possible to obtain overlapping sequences demonstrating the uniqueness of the BglII, ClaI and PstI restriction sites and making it possible to identify the probable ATG for initiation of translation of the K. lactis PRB1 gene.

E.1.7. Nucleotide sequence of the K. lactis PRB1 gene.

The compilation of the sequences determined in E.1.4., E.1.5 and E.1.6. covers the entire coding phase of the K. lactis PRB1 gens (FIG. 6, panel D). This sequence is given SEQ ID No. 1 and encodes a protein of 561 residues corresponding to the K. lactis protease B.

Example 2 CLONING OF K. LACTIS CARBOXYPEPTIDASE Y GENE

The general strategy described in Example 1 is repeated for the cloning of the carboxypeptidase Y gene of K. lactis CBS 2359/152.

E2.1. Preparation of the probe.

A preparation of genomic DNA of the strain S. cerevisiae S288C [Mortimer and Johnston, Genetics 113 (1986) 35] is first carried out according to Example E1.2. A PCR amplification of this genomic DNA preparation is then carried out with the oligonucleotides 5′-CTTCTTGGAGTT GTTCTTCG-3′ and 5′-TGGCAAGACATCC GTCCACGCCTTATT-ACC-3′, Specific for the PRC1 gene. An amplified fragment of the expected size (699 bp) is thus obtained which corresponds to positions 696-1395 (the ATG initiation codon being numbered +1) of the open reading frame of the S. Cerevisiae PRC1 gens [Valls et al., Cell 48 (1987) 887]. This fragment is then purified by electroelution and radiolabelled according to the “Random Priming” technique.

E.2.2. Cloning of the K. lactis PRC1 gene.

The K. lactis PRC1 gene is obtained by screening the K. lactis genomic library constructed Wésolowski-Louvel [Yeast A (1988) 71] from the strain 2359/152 in the cloning vector KEp6 [Chen et al., J. Basic. Microbiol. 28 (1988) 211]. The stain E. coli JM101 is transformed with the DNA from the library and the transformants are plated on LB medium supplemented with ampicillin (50 mg/l). 15,000 clones are then transferred onto nitrocellulose filters and the filters are hybridized with the probe described in Example E.2.1. The hybridization and washing conditions are those of Example E.1.3. 12 positive clones are thus isolated and one of them, designated pC34, is selected for the rest of the study. In a first instance, the hybridization of the plasmid pC34 with the probe corresponding to the S. cerevisiae PRC1 gene is confirmed by Southern blotting. A restriction map of the genomic insert (about 6.9 kb)of the plasmid pC34 is given in FIG. 7. The sequence of the 2.5 kb EcoRI-SalI fragment comprising the K. lactis PRC1 gene is then determined on the 2 strands. This sequence is presented SEQ ID No.3.

Example 3 CLONlNG OF THE K. LACTIS PROTEASE A GENE

The general strategy described in the preceding examples is repeated for the cloning of the protease A gene from K. lactis CBS 2359/152.

E.3.1. Preparation of the probe.

An inner fragment of 449 bp of the PRA1 gene (or PEP4 gene) from S. cerevisiae is first amplified by the PCR technique starting with the plasmid CBZIB1 [Woolford et al., Mol. Cell. Biol. 6 (1986) 2500] provided by Dr E. Jones (Carnegie-Mellon University, Pittsburgh, Pa., USA) and the oligodeoxynucleotides 5′-CTGTTGATAAGGTGGTCC-3′ (SEQ ID NO. 15) and 5′CAAGCGTGTAATCGTATGGC-3′ (SEQ ID NO. 16). The amplified fragment obtained corresponds to positions 617-1066 of the open reading frame of the S. cerevisiae PRA1 gene, the ATG initiation codon being numbered +1. This fragment is then purified by electroelution and radiolabelled according to the “Random Priming” technique.

E3.2. Cloning of the K. lactis PRA1 gene.

The PRA1 gene is obtained by screening the K. lactis genomic library constructed by Wéselowski-Louvel [Yeast 4 (1988) 71] starting with the strain 2359/152 in the cloning vector KEp6 [Chen et al., J. Basic Microbiol. 28 (1988) 211]. After transforming the library in E. coli JM101 and selecting in the presence of ampicillin (50 mg/l). 15,000 clones are then transferred onto nitrocellulose filters and the filters are hybridized with the probe described in Example E3.1. The hybridization and washing conditions are those of Example E.1.3. Only 1 positive clone was thus isolated and designated pA25/1. In a first instance, the hybridization of the plasmid pA25/1 with the probe corresponding to the S. cerevisiae PRA1 gene is confirmed by Southern blotting. A restriction map of the genomic insert (about 7.5 kb) of this plasmid is represented in FIG. 8. The sequence of the 1.6 kb ClaI-EcoRI fragment comprising the K. lactis PRA1 gene is then determined on the 2 strands. This sequence is presented SEQ ID No. 5.

Example 4 TRANSFORMATION OF THE YEASTS

The transformation of the yeasts belonging to the genus Kluyveromyces, and in particular the strains K. lactis MW98-8C, CBS 293.91 and CBS 294.91 (uraA) is carried out for example by the technique for treating whole cells with lithium acetate [Ito H. et al., J. Bacteriol. 153 (1983) 163-168], modified as follows. The growth of the cells occurs at 28° C. in 50 ml of YPD medium, with stirring and up to an optical density of 600 nm (OD600) of between 0.6 and 0.8; the cells are then harvested by low-speed centrifugation, washed in a sterile solution of TE (10 mMTris HCl pH 7.4; 1 mM EDTA), resuspended in 3-4 ml of lithium acetate (0.1 M in TE) in order to obtain a cell density of about 2×10⁸ cells/ml, and then incubated at 30° C. for 1 hour with gentle stirring. 0.1 ml aliquots of the resulting suspension of competent cells are incubated at 30° C. for 1 hour in the presence of DNA and at a final concentration of 35% polyethylene glycol (PEG₄₀₀₀, Sigma). After a heat shock of 5 minutes at 42° C., the cells are washed twice, then resuspended in 0.2 ml sterile water. In the case or the selectable marker is the S. cerevisiae URA3 gene, the cells are directly plated on YNB (Yeast Nitrogen Base; Difco)/glucose (20 g/l)/agar. In the case or the selectable marker is the aph gene of the transposon Tn903, the cells are first incubated for 16 hours at 28° C. in 2 ml of YPD medium so as to allow the phenotypic expression of the G418 resistance gene expressed under the control of the P_(k1) promoter (cf. EP 361 991); 200 μl of the cellular suspension are then plated on selective YPD dishes (G418, 200 μg/ml). The dishes are incubated at 28° C. and the disruptants or the transformants appear after 2 to 3 days of cell growth.

Example 5 DISRUPTION OF PROTEASE GENES IN K. LACTIS

E.5.1. K. lactis strains disrupted for the PRB1 gene.

The plasmid pYG1229 is constructed by cloning the HindIII-EcoRI fragment (including the BglII-EcoRI fragment of about 700 bp and corresponding to the C-terminal part of the K. Lactis PRB1 gene) of the plasmid pYG1224 between the corresponding sites of the plasmid pUC9. The plasmid pYG1228 is constructed by cloning the HindIII fragment of 1.1 kb and corresponding to the S. cerevisiae URA3 gens derived from the plasmid pCG3 [Gerbaud et al., Curt. Genetics A (1981) 173] in the HindIII site of the plasmid pIC-20R. The plasmid pYG1228 therefore makes it possible to have a SalI-XhoI restriction fragment of about 1.1 kb and containing the entire HindIII fragment containing the S. cerevisiae URA3 gens. This restriction fragment is then cloned into the SalI site of the plasmid pYG1229 which generates the plasmid pYG1232 (2 possible orientations). The digestion of this plasmid with the BglII and EcoRI enzymes makes it possible to generate a restriction fragment of about 1.8 kb corresponding to the S. Cerevisiae URA3 gene bordered by K. lactis genomic sequences derived from the PRB1 gene (FIG. 9, panel A). The transformation of the K. lactis uraA mutants with the BglII-EcoRI fragment of the plasmid pYG1232 generates transformed clones (complemented by the S. cerevisiae URA3 gens) corresponding to the integration of this fragment in to the chromosome. Panel B of FIG. 9 shows the integration of this fragment into the genomic DNA of the K. lactis CBS 294.91 strain (uraA) after non-homologous recombination (well 1) or after homologous recombination in the PRB1 gene (well 3, this disruptant is noted Y750). The disruption of the wild-type allel of the PRB1 gene does not modify the growth characteristics of the strain.

E.5.2. K. lactis strains disrupted for the PRC1 gene.

The 4.4 kb SalI-SphI fragment derived from the plasmid pC34 is first subcloned into the corresponding sites of the vector pIC-20R, which generates the plasmid pYG154 (FIG. 12, panel b). This plasmid is then digested with the SphI enzyme, then treated with phage T4 DNA polymerase I in the presence of calf intestinal phosphatase (CIP). The plasmid obtained is then ligated to the EcoRI fragment of 1.6 kb carrying the S. cerevisiae URA3 gene derived from the plasmid pKan707 (EP 361 991), previously treated with the Klenow fragment of DNA polymerase I of E. coli. The plasmid obtained is designated pYG155 (FIG. 10, panel c). The 4.5 kb SalI-BamHI fragment of the plasmid pYG155 is then purified by electroelution and used to transform the K. lactis CBS 294.91 strain (uraA). The transformants are selected for the Ura⁺ phenotype and a few clones are then analysed by Southern blotting in order to check the site of integration of the URA3 marker. The clone Y797 is thus identified in which the chromosomal PRC1 gene has been replaced, by homologous recombination, by the disrupted allel constructed in vitro. The disruption of the wild-type allele of the PRC1 gene does not modify the growth characteristics of the strain.

Example 6 EXPRESSION PLASMIDS

E.6.1. Plasmid pYG1212.

The genes for the proteins of interest which it is desired to secrete and/or express are first inserted, “in the productive orientation” (defined as the orientation which places the N-terminal region of the protein proximally relative to the transcription promoter), under the control of regulatable or constitutive functional promoters such as for example those present in the plasmids pYG105 (K- lactis LAC4 promoter), pYG106 (S. cerevisiae PGK promoter), pYG536 (S. cerevisiae PHO5 promoter), or hybrid promoters such as those described in patent application EP 361 991. The plasmids pYG105 and pYG106 are particularly useful because they allow the expression of genes included in HindIII restriction fragments from regulatable (pYG105) or constitutive (pYG106) promoters which are functional in K. lactis.

The plasmid pYG105 corresponds to the plasmid pKan707 described in patent application EP 361 991 in which the unique HindIII restriction site located in the gene for resistance to geneticin (G418) has been destroyed by site-directed mutagenesis, conserving a protein unchanged (oligodeoxynucleotide 5′GAAATGCATAAGCT CTTGCCATTCTCACCG-3′) (SEQ ID NO. 17). The SalI-SacI fragment encoding the URA3 gene of the plasmid thus mutated was then replaced by a Sail-Sac1 restriction fragment containing an expression cassette consisting of the K. lactis LAC4 promoter [in the form of a SalI-HindIII fragment derived from the plasmid pYG1075 Fleer et al., Bio/Technology 9 (1991) 968]) and the S. cerevisiae PGK gene terminator [in the form of a HindIII-SacI fragment; Fleer et al., Bio/Technology 9 (1991) 968]. The plasmid pYG105 is mitotically very stable in Kluyveromyces yeasts and a restriction map is given in FIG. 11. The plasmids pYG105 and pYG106 differ from each other only in the nature of the transcription promoter encoded by the SalI-HindIII fragment.

The protein encoded by the plasmid pYG1212 corresponds approximately to the first two domains of human serum albumin (HSA). This molecular variant, obtained by digestion of the C-terminal end of HSA using exonuclease Ba131 from the unique MstII site located 3 amino acids from the C-terminal end of HSA, is derived from the plasmid YP40 described in patent application EP 413 622. Briefly, the HindIII-MstII restriction fragment of the plasmid YP40, corresponding to residues 1 to 403 of RSA (the ATG for initiation of translation is noted +1), is ligated to the MstII-HindIII fragment of the plasmid pYG221 [Yeh et al., Proc. Natl. Acad. Sci. USA 89 (1992) 1904], which generates a HindIII fragment including the 403 N-terminal residues of HSA followed by the last three residues of HSA (residues Leu-Gly-Leu) and a translational stop codon [truncated variant noted HSA₍₁₋₄₀₃₎]. The HindIII fragment is then cloned in the productive orientation into the plasmid pYG105, which generates the plasmid pYG1212 (FIG. 11).

Example 7 SECRETION POTENTIAL OF THE DISRUPTANTS

E.7.1. HSA₍₁₋₄₀₃₎ in a disruptant for protease B.

In a first stage, the yeasts K. Lactis CS 293.91 and Y750 are transformed with the plasmid pYG1212. After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the protein HSA₍₁₋₄₀₃₎. A few clones are incubated in YPD or YPL medium at 28° C. The culture supernatents are recovered by centrifugation when the cells reach the stationary growth phase, concentrated 10 fold by precipitation for 30 minutes at −20° C. in a final concentration of 60% ethanol, then tested after electrophoresis on an 8.5% SDS-PAGE gel and staining of the gel with coomassie blue. The results presented in FIG. 12 demonstrate that the disruptant Y750 (prb1°) secretes quantities of protein which are much higher than the quantities secreted by its non-disrupted homologue. This is true after 2, 4 or 7 days of growth, independently of the carbon source used (glucose or lactose).

17 1685 base pairs nucleic acid double linear cDNA Kluyveromyces lactis CDS 1..1683 /product= “Protease B gene” /gene= “K1.PRB1” 1 ATG AAG TTC GAA AAT ACA TTA TTG ACT ATA ACC GCA TTG TCT ACC GTG 48 Met Lys Phe Glu Asn Thr Leu Leu Thr Ile Thr Ala Leu Ser Thr Val 1 5 10 15 GCT ACT GCT TTG GTT ATC CCT GAA GTT AAT AGG GAA AAC AAG CAT GGT 96 Ala Thr Ala Leu Val Ile Pro Glu Val Asn Arg Glu Asn Lys His Gly 20 25 30 GAC AAG AGC GTT GCC ATC AAA GAT CAT GCT TCT TCT GAT TTG GAT AAG 144 Asp Lys Ser Val Ala Ile Lys Asp His Ala Ser Ser Asp Leu Asp Lys 35 40 45 CCT CAA CAT CAT GCT AAT GGC AAG GCT CGT TCT AAG TCT CGT GGT CGC 192 Pro Gln His His Ala Asn Gly Lys Ala Arg Ser Lys Ser Arg Gly Arg 50 55 60 TGC GCA GAC TCC AAG AAA TTC GAC AAG CTA CGT CCA GTC GAC GAT GCT 240 Cys Ala Asp Ser Lys Lys Phe Asp Lys Leu Arg Pro Val Asp Asp Ala 65 70 75 80 TCA GCT ATT TTA GCT CCA CTT TCT ACA GTT AAT GAT ATT GCC AAC AAG 288 Ser Ala Ile Leu Ala Pro Leu Ser Thr Val Asn Asp Ile Ala Asn Lys 85 90 95 ATT CCT AAT CGT TAC ATC ATT GTC TTT AAG AAA GAT GCC TCT GCA GAT 336 Ile Pro Asn Arg Tyr Ile Ile Val Phe Lys Lys Asp Ala Ser Ala Asp 100 105 110 GAA GTG AAG TTC CAT CAA GAA CTA GTC TCT GTC GAA CAT GCC AAG GCA 384 Glu Val Lys Phe His Gln Glu Leu Val Ser Val Glu His Ala Lys Ala 115 120 125 CTA GGT TCC TTA GCT GAC CAT GAC CCA TTC TTC ACA GCA ACT TCC GGT 432 Leu Gly Ser Leu Ala Asp His Asp Pro Phe Phe Thr Ala Thr Ser Gly 130 135 140 GAA CAT AGT GAA TTT GGT GTC AAA GCA CAC TCT TTG GAA GGT GGT ATT 480 Glu His Ser Glu Phe Gly Val Lys Ala His Ser Leu Glu Gly Gly Ile 145 150 155 160 CAA GAC TCT TTT GAT ATT GCC GGT TCC CTT TCT GGT TAT GTT GGC TAC 528 Gln Asp Ser Phe Asp Ile Ala Gly Ser Leu Ser Gly Tyr Val Gly Tyr 165 170 175 TTC ACA AAA GAA GTT ATC GAT TTC ATC AGA AGA AGC CCA TTG GTT GAA 576 Phe Thr Lys Glu Val Ile Asp Phe Ile Arg Arg Ser Pro Leu Val Glu 180 185 190 TTT GTT GAA GAA GAT TCT ATG GTT TTC TCT AAT AGT TTC AAT ACC CAA 624 Phe Val Glu Glu Asp Ser Met Val Phe Ser Asn Ser Phe Asn Thr Gln 195 200 205 AAC AGT GCT CCT TGG GGT CTA GCT CGT ATT TCT CAT CGT GAA AAG TTG 672 Asn Ser Ala Pro Trp Gly Leu Ala Arg Ile Ser His Arg Glu Lys Leu 210 215 220 AAT TTA GGA TCT TTC AAC AAG TAC TTG TAT GAT GAT GAC GCT GGT AAA 720 Asn Leu Gly Ser Phe Asn Lys Tyr Leu Tyr Asp Asp Asp Ala Gly Lys 225 230 235 240 GGT GTT ACT GCT TAC GTT GTT GAC ACT GGT GTC AAT GTC AAC CAT AAG 768 Gly Val Thr Ala Tyr Val Val Asp Thr Gly Val Asn Val Asn His Lys 245 250 255 GAC TTT GAT GGC AGA GCT GTT TGG GGT AAG ACT ATT CCA AAA GAT GAT 816 Asp Phe Asp Gly Arg Ala Val Trp Gly Lys Thr Ile Pro Lys Asp Asp 260 265 270 CCA GAT GTA GAT GGA AAT GGT CAC GGT ACC CAC TGT GCT GGT ACC ATC 864 Pro Asp Val Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Ile 275 280 285 GGT TCG GTT CAT TAT GGT GTT GCT AAG AAT GCT GAT ATA GTT GCC GTT 912 Gly Ser Val His Tyr Gly Val Ala Lys Asn Ala Asp Ile Val Ala Val 290 295 300 AAG GTT TTG AGA TCT AAT GGT TCT GGT ACC ATG TCT GAT GTT GTT AAA 960 Lys Val Leu Arg Ser Asn Gly Ser Gly Thr Met Ser Asp Val Val Lys 305 310 315 320 GGT GTC GAA TAT GTT GCC GAA GCA CAC AAG AAA GCT GTT GAA GAA CAA 1008 Gly Val Glu Tyr Val Ala Glu Ala His Lys Lys Ala Val Glu Glu Gln 325 330 335 AAG AAA GGG TTC AAG GGT TCA ACT GCT AAC ATG TCT TTG GGT GGT GGT 1056 Lys Lys Gly Phe Lys Gly Ser Thr Ala Asn Met Ser Leu Gly Gly Gly 340 345 350 AAA TCT CCA GCC TTG GAT TTG GCC GTC AAC GCC GCT GTT AAG GCA GGT 1104 Lys Ser Pro Ala Leu Asp Leu Ala Val Asn Ala Ala Val Lys Ala Gly 355 360 365 GTT CAT TTT GCT GTT GCT GCC GGT AAT GAG AAC CAA GAT GCT TGT AAC 1152 Val His Phe Ala Val Ala Ala Gly Asn Glu Asn Gln Asp Ala Cys Asn 370 375 380 ACT TCG CCT GCC GCG GCT GAG AAT GCT ATC ACG GTT GGT GCC TCC ACA 1200 Thr Ser Pro Ala Ala Ala Glu Asn Ala Ile Thr Val Gly Ala Ser Thr 385 390 395 400 TTA AGT GAT GAA AGA GCT TAC TTT TCC AAT TGG GGT AAA TGT GTC GAC 1248 Leu Ser Asp Glu Arg Ala Tyr Phe Ser Asn Trp Gly Lys Cys Val Asp 405 410 415 ATC TTT GGT CCG GGT TTG AAT ATC TTA TCT ACC TAC ATT GGT TCT GAT 1296 Ile Phe Gly Pro Gly Leu Asn Ile Leu Ser Thr Tyr Ile Gly Ser Asp 420 425 430 ACT GCT ACT GCT ACC TTG TCT GGT ACT TCT ATG GCC ACT CCT CAT GTT 1344 Thr Ala Thr Ala Thr Leu Ser Gly Thr Ser Met Ala Thr Pro His Val 435 440 445 GTC GGT TTG CTA ACA TAT TTC TTG TCC TTG CAA CCA GAT GCT GAT AGT 1392 Val Gly Leu Leu Thr Tyr Phe Leu Ser Leu Gln Pro Asp Ala Asp Ser 450 455 460 GAA TAT TTC CAT GCC GCT GGC GGT ATT ACT CCT TCC CAA CTC AAG AAG 1440 Glu Tyr Phe His Ala Ala Gly Gly Ile Thr Pro Ser Gln Leu Lys Lys 465 470 475 480 AAG TTA ATT GAT TTC TCT ACT AAG AAC GTA TTG TCC GAT CTA CCT GAA 1488 Lys Leu Ile Asp Phe Ser Thr Lys Asn Val Leu Ser Asp Leu Pro Glu 485 490 495 GAT ACC GTG AAC TAC TTG ATT TAC AAC GGT GGT GGT CAA GAT TTG GAT 1536 Asp Thr Val Asn Tyr Leu Ile Tyr Asn Gly Gly Gly Gln Asp Leu Asp 500 505 510 GAC CTA TGG GGT AAG GAT TAC TCT ATT GGA AAA GAA CCA TCT GCC AAC 1584 Asp Leu Trp Gly Lys Asp Tyr Ser Ile Gly Lys Glu Pro Ser Ala Asn 515 520 525 CCT GAA TTC AGC TTG GAA AGC TTG ATT AAC TCT TTG GAT TCA AAG ACT 1632 Pro Glu Phe Ser Leu Glu Ser Leu Ile Asn Ser Leu Asp Ser Lys Thr 530 535 540 GAT GCT ATC TTT GAC GAC GTT AGA CAG TTG TTG GAC CAA TTT AAT ATC 1680 Asp Ala Ile Phe Asp Asp Val Arg Gln Leu Leu Asp Gln Phe Asn Ile 545 550 555 560 ATC TA 1685 Ile 561 amino acids amino acid linear protein not provided 2 Met Lys Phe Glu Asn Thr Leu Leu Thr Ile Thr Ala Leu Ser Thr Val 1 5 10 15 Ala Thr Ala Leu Val Ile Pro Glu Val Asn Arg Glu Asn Lys His Gly 20 25 30 Asp Lys Ser Val Ala Ile Lys Asp His Ala Ser Ser Asp Leu Asp Lys 35 40 45 Pro Gln His His Ala Asn Gly Lys Ala Arg Ser Lys Ser Arg Gly Arg 50 55 60 Cys Ala Asp Ser Lys Lys Phe Asp Lys Leu Arg Pro Val Asp Asp Ala 65 70 75 80 Ser Ala Ile Leu Ala Pro Leu Ser Thr Val Asn Asp Ile Ala Asn Lys 85 90 95 Ile Pro Asn Arg Tyr Ile Ile Val Phe Lys Lys Asp Ala Ser Ala Asp 100 105 110 Glu Val Lys Phe His Gln Glu Leu Val Ser Val Glu His Ala Lys Ala 115 120 125 Leu Gly Ser Leu Ala Asp His Asp Pro Phe Phe Thr Ala Thr Ser Gly 130 135 140 Glu His Ser Glu Phe Gly Val Lys Ala His Ser Leu Glu Gly Gly Ile 145 150 155 160 Gln Asp Ser Phe Asp Ile Ala Gly Ser Leu Ser Gly Tyr Val Gly Tyr 165 170 175 Phe Thr Lys Glu Val Ile Asp Phe Ile Arg Arg Ser Pro Leu Val Glu 180 185 190 Phe Val Glu Glu Asp Ser Met Val Phe Ser Asn Ser Phe Asn Thr Gln 195 200 205 Asn Ser Ala Pro Trp Gly Leu Ala Arg Ile Ser His Arg Glu Lys Leu 210 215 220 Asn Leu Gly Ser Phe Asn Lys Tyr Leu Tyr Asp Asp Asp Ala Gly Lys 225 230 235 240 Gly Val Thr Ala Tyr Val Val Asp Thr Gly Val Asn Val Asn His Lys 245 250 255 Asp Phe Asp Gly Arg Ala Val Trp Gly Lys Thr Ile Pro Lys Asp Asp 260 265 270 Pro Asp Val Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Ile 275 280 285 Gly Ser Val His Tyr Gly Val Ala Lys Asn Ala Asp Ile Val Ala Val 290 295 300 Lys Val Leu Arg Ser Asn Gly Ser Gly Thr Met Ser Asp Val Val Lys 305 310 315 320 Gly Val Glu Tyr Val Ala Glu Ala His Lys Lys Ala Val Glu Glu Gln 325 330 335 Lys Lys Gly Phe Lys Gly Ser Thr Ala Asn Met Ser Leu Gly Gly Gly 340 345 350 Lys Ser Pro Ala Leu Asp Leu Ala Val Asn Ala Ala Val Lys Ala Gly 355 360 365 Val His Phe Ala Val Ala Ala Gly Asn Glu Asn Gln Asp Ala Cys Asn 370 375 380 Thr Ser Pro Ala Ala Ala Glu Asn Ala Ile Thr Val Gly Ala Ser Thr 385 390 395 400 Leu Ser Asp Glu Arg Ala Tyr Phe Ser Asn Trp Gly Lys Cys Val Asp 405 410 415 Ile Phe Gly Pro Gly Leu Asn Ile Leu Ser Thr Tyr Ile Gly Ser Asp 420 425 430 Thr Ala Thr Ala Thr Leu Ser Gly Thr Ser Met Ala Thr Pro His Val 435 440 445 Val Gly Leu Leu Thr Tyr Phe Leu Ser Leu Gln Pro Asp Ala Asp Ser 450 455 460 Glu Tyr Phe His Ala Ala Gly Gly Ile Thr Pro Ser Gln Leu Lys Lys 465 470 475 480 Lys Leu Ile Asp Phe Ser Thr Lys Asn Val Leu Ser Asp Leu Pro Glu 485 490 495 Asp Thr Val Asn Tyr Leu Ile Tyr Asn Gly Gly Gly Gln Asp Leu Asp 500 505 510 Asp Leu Trp Gly Lys Asp Tyr Ser Ile Gly Lys Glu Pro Ser Ala Asn 515 520 525 Pro Glu Phe Ser Leu Glu Ser Leu Ile Asn Ser Leu Asp Ser Lys Thr 530 535 540 Asp Ala Ile Phe Asp Asp Val Arg Gln Leu Leu Asp Gln Phe Asn Ile 545 550 555 560 Ile 2503 base pairs nucleic acid double linear cDNA Kluyveromyces lactis CDS 387..1862 /product= “K. lactis protease C gene” /gene= “K1.PRC1” 3 GAATTCTGTC AACTGGATAC GGAAGACAAT AGAATGGACA CATAATGGTC TCAATACGAC 60 AATTCAACGG CTCTTAGAAG GTGAGTTATT CTTGACATTT TCATGGCTCT TCGAGCATGC 120 TTTCTAAGAT GACGCGGAAG GTGAAAAAGA TTAGAAAACG GCCATTCACG TGAATATCAC 180 GTGAACTACA AATTCATGAT ATATTACCGC CAATAGTATT GGTGGTTACC CGATCGTATC 240 GAATGTACTG ACTTCGAAAA TATGAATAGT CCTCTTTAAA ACAAAGGGTT TTCAGTGACC 300 CTTACTCCAT CATCTCCTTA GTATTTGGTC TACAGACTCG CCATTGCCGT ATATTCAGGG 360 TAGTAGTCAG TACATCGGTG TCTGCC ATG GTT TCG ATA AAG TTT CTT TTA TCT 413 Met Val Ser Ile Lys Phe Leu Leu Ser 1 5 TTA TAC GGC TGG CTA TCT GTC ACT TTA GCC ATC TCG TTG AAT GCC GTT 461 Leu Tyr Gly Trp Leu Ser Val Thr Leu Ala Ile Ser Leu Asn Ala Val 10 15 20 25 GTT GAT AGT TTA TTC TCG AAC AGT TTC GAC GGG AAT AAC AAC ATC GAG 509 Val Asp Ser Leu Phe Ser Asn Ser Phe Asp Gly Asn Asn Asn Ile Glu 30 35 40 GAT CAT GAA ACT GCA AAT TAT AAC ACT CAG TTT AGT GTC TTC AGC TCA 557 Asp His Glu Thr Ala Asn Tyr Asn Thr Gln Phe Ser Val Phe Ser Ser 45 50 55 AAT ATT GAC GAC GCT TAT TCA TTG AGA ATT AAA CCT TTG GAT CCC AAA 605 Asn Ile Asp Asp Ala Tyr Ser Leu Arg Ile Lys Pro Leu Asp Pro Lys 60 65 70 TCT CTT GGC GTT GAT ACC GTG AAA CAA TGG TCG GGA TAT TTA GAT TAC 653 Ser Leu Gly Val Asp Thr Val Lys Gln Trp Ser Gly Tyr Leu Asp Tyr 75 80 85 CAG GAC TCA AAA CAC TTC TTT TAT TGG TTT TTT GAG TCT AGA AAT GAC 701 Gln Asp Ser Lys His Phe Phe Tyr Trp Phe Phe Glu Ser Arg Asn Asp 90 95 100 105 CCA GAG AAT GAC CCA GTG ATA CTA TGG TTA AAC GGT GGT CCT GGC TGT 749 Pro Glu Asn Asp Pro Val Ile Leu Trp Leu Asn Gly Gly Pro Gly Cys 110 115 120 TCC TCT TTC GTC GGT CTT TTC TTT GAA TTG GGA CCT TCT TCT ATA GGA 797 Ser Ser Phe Val Gly Leu Phe Phe Glu Leu Gly Pro Ser Ser Ile Gly 125 130 135 GCT GAT TTG AAA CCC ATT TAT AAC CCC TAC TCT TGG AAT TCC AAC GCT 845 Ala Asp Leu Lys Pro Ile Tyr Asn Pro Tyr Ser Trp Asn Ser Asn Ala 140 145 150 TCT GTG ATA TTC CTA GAT CAG CCT GTT GGT GTT GGG TTC TCA TAC GGT 893 Ser Val Ile Phe Leu Asp Gln Pro Val Gly Val Gly Phe Ser Tyr Gly 155 160 165 GAC TCT AAA GTG TCT ACT ACA GAT GAC GCT GCC AAA GAC GTT TAC ATA 941 Asp Ser Lys Val Ser Thr Thr Asp Asp Ala Ala Lys Asp Val Tyr Ile 170 175 180 185 TTC TTA GAT TTG TTC TTT GAA AGA TTC CCT CAT TTG AGA AAT AAC GAT 989 Phe Leu Asp Leu Phe Phe Glu Arg Phe Pro His Leu Arg Asn Asn Asp 190 195 200 TTC CAT ATC TCC GGT GAA TCA TAC GCC GGT CAT TAT TTA CCC AAG ATT 1037 Phe His Ile Ser Gly Glu Ser Tyr Ala Gly His Tyr Leu Pro Lys Ile 205 210 215 GCT CAT GAG ATT GCT GTA GTG CAT GCT GAG GAT TCC TCC TTC AAT CTA 1085 Ala His Glu Ile Ala Val Val His Ala Glu Asp Ser Ser Phe Asn Leu 220 225 230 TCG TCA GTA TTA ATT GGA AAT GGA TTT ACT GAC CCA CTG ACT CAA TAC 1133 Ser Ser Val Leu Ile Gly Asn Gly Phe Thr Asp Pro Leu Thr Gln Tyr 235 240 245 CAA TAT TAC GAG CCG ATG GCC TGT GGT GAA GGT GGT TAT CCA GCG GTG 1181 Gln Tyr Tyr Glu Pro Met Ala Cys Gly Glu Gly Gly Tyr Pro Ala Val 250 255 260 265 TTG GAA CCG GAA GAT TGC TTA GAT ATG AAT AGG AAT CTA CCT CTA TGC 1229 Leu Glu Pro Glu Asp Cys Leu Asp Met Asn Arg Asn Leu Pro Leu Cys 270 275 280 CTA TCG CTT GTG GAC CGC TGT TAC AAG TCC CAT TCT GTT TTC TCT TGT 1277 Leu Ser Leu Val Asp Arg Cys Tyr Lys Ser His Ser Val Phe Ser Cys 285 290 295 GTG TTG GCT GAC CGT TAT TGT GAA CAA CAG ATT ACT GGG GTT TAT GAG 1325 Val Leu Ala Asp Arg Tyr Cys Glu Gln Gln Ile Thr Gly Val Tyr Glu 300 305 310 AAA TCA GGT AGG AAC CCT TAC GAT ATT AGA TCT AAG TGT GAA GCA GAG 1373 Lys Ser Gly Arg Asn Pro Tyr Asp Ile Arg Ser Lys Cys Glu Ala Glu 315 320 325 GAT GAT TCC GGT GCC TGT TAT CAG GAA GAA ATT TAT ATC TCT GAT TAC 1421 Asp Asp Ser Gly Ala Cys Tyr Gln Glu Glu Ile Tyr Ile Ser Asp Tyr 330 335 340 345 TTG AAT CAG GAG GAA GTT CAA AGA GCT TTA GGG ACT GAT GTG AGT TCT 1469 Leu Asn Gln Glu Glu Val Gln Arg Ala Leu Gly Thr Asp Val Ser Ser 350 355 360 TTC CAA GGT TGT AGC TCG GAT GTC GGT ATC GGT TTC GCA TTC ACT GGC 1517 Phe Gln Gly Cys Ser Ser Asp Val Gly Ile Gly Phe Ala Phe Thr Gly 365 370 375 GAT GGA CCG AGC CCA TTC CAC CAG TAC GTC GCA GAA CTT CTT GAT CAA 1565 Asp Gly Pro Ser Pro Phe His Gln Tyr Val Ala Glu Leu Leu Asp Gln 380 385 390 GAT ATC AAT GTC TTG ATA TAT GCA GGC GAT AAG GAT TAT ATT TGT AAT 1613 Asp Ile Asn Val Leu Ile Tyr Ala Gly Asp Lys Asp Tyr Ile Cys Asn 395 400 405 TGG CTA GGA AAT CTC GCT TGG ACT GAA AAA TTG GAA TGG AGG TAT AAC 1661 Trp Leu Gly Asn Leu Ala Trp Thr Glu Lys Leu Glu Trp Arg Tyr Asn 410 415 420 425 GAA GAG TAT AAA AAA CAA GTT TTG AGA ACT TGG AAG AGT GAA GAA ACA 1709 Glu Glu Tyr Lys Lys Gln Val Leu Arg Thr Trp Lys Ser Glu Glu Thr 430 435 440 GAT GAG ACC ATT GGC GAA ACC AAA TCT TAT GGC CCG CTA ACT TAC TTG 1757 Asp Glu Thr Ile Gly Glu Thr Lys Ser Tyr Gly Pro Leu Thr Tyr Leu 445 450 455 AGA ATC TAT GAT GCT GGA CAC ATG GTT CCT CAC GAC CAA CCT GAA AAT 1805 Arg Ile Tyr Asp Ala Gly His Met Val Pro His Asp Gln Pro Glu Asn 460 465 470 TCA TTA CAA ATG GTG AAT TCA TGG ATT CAG AAT ATC GCA AAG AGA TCT 1853 Ser Leu Gln Met Val Asn Ser Trp Ile Gln Asn Ile Ala Lys Arg Ser 475 480 485 AGA ATA TAAGCATATT TCTTTACAAT TAATTTTAAA TACAAGCACC CTGAGGTATA 1909 Arg Ile 490 TACTGTATGC AGTTTGTTGC ATATCTATCA TTTCTTTCGC AATTGTTCAC TTTTGATTCA 1969 TTCTGTACAC TCTAATAAGG TTTTGCAACC TAGTAATGAT TTCCACACAT TCTTCAGCCG 2029 ACACAGCTTC GAAATAATAT CTCCGTTCTC TATCAGGTCT GTGAACAAAA ATCTTGAAAT 2089 ATTCTGGAAC GCGCTTAGAC CTTTTCACCA GGGTAATTTG GCTTATATGG AACGATTTCG 2149 TCTTGACGTT TTCGTGTGTC CAATTGAAAT CACCATCAGG CCCACTTATA TAAACGTAGT 2209 CACCATCAAT GACCAACATC CTCTCGTGTT TATTGATGAA TGACATTTGT TGTCTTCTCC 2269 ATACCTTATA TTTATAGTAG GAACCAGCAA AGAGATCTTT GTAACTGTTA TTTCCAGAAA 2329 CGTTATTTGA TGGTTTGGAT AAACTCCCAA GTTTAGGAGA TTTCTGGCCA TTGTTGAGAG 2389 AAGATTTTGA GGAATTTTTG AGTTTAAAAA AGCCAGAGGT AGAGGAAGAT GTGTTATGCT 2449 GCTTGTTGAT ACTGAATAAA TTCTTTGAGC TTGTTCTGTT CGAGGTTGGT CGAC 2503 491 amino acids amino acid linear protein not provided 4 Met Val Ser Ile Lys Phe Leu Leu Ser Leu Tyr Gly Trp Leu Ser Val 1 5 10 15 Thr Leu Ala Ile Ser Leu Asn Ala Val Val Asp Ser Leu Phe Ser Asn 20 25 30 Ser Phe Asp Gly Asn Asn Asn Ile Glu Asp His Glu Thr Ala Asn Tyr 35 40 45 Asn Thr Gln Phe Ser Val Phe Ser Ser Asn Ile Asp Asp Ala Tyr Ser 50 55 60 Leu Arg Ile Lys Pro Leu Asp Pro Lys Ser Leu Gly Val Asp Thr Val 65 70 75 80 Lys Gln Trp Ser Gly Tyr Leu Asp Tyr Gln Asp Ser Lys His Phe Phe 85 90 95 Tyr Trp Phe Phe Glu Ser Arg Asn Asp Pro Glu Asn Asp Pro Val Ile 100 105 110 Leu Trp Leu Asn Gly Gly Pro Gly Cys Ser Ser Phe Val Gly Leu Phe 115 120 125 Phe Glu Leu Gly Pro Ser Ser Ile Gly Ala Asp Leu Lys Pro Ile Tyr 130 135 140 Asn Pro Tyr Ser Trp Asn Ser Asn Ala Ser Val Ile Phe Leu Asp Gln 145 150 155 160 Pro Val Gly Val Gly Phe Ser Tyr Gly Asp Ser Lys Val Ser Thr Thr 165 170 175 Asp Asp Ala Ala Lys Asp Val Tyr Ile Phe Leu Asp Leu Phe Phe Glu 180 185 190 Arg Phe Pro His Leu Arg Asn Asn Asp Phe His Ile Ser Gly Glu Ser 195 200 205 Tyr Ala Gly His Tyr Leu Pro Lys Ile Ala His Glu Ile Ala Val Val 210 215 220 His Ala Glu Asp Ser Ser Phe Asn Leu Ser Ser Val Leu Ile Gly Asn 225 230 235 240 Gly Phe Thr Asp Pro Leu Thr Gln Tyr Gln Tyr Tyr Glu Pro Met Ala 245 250 255 Cys Gly Glu Gly Gly Tyr Pro Ala Val Leu Glu Pro Glu Asp Cys Leu 260 265 270 Asp Met Asn Arg Asn Leu Pro Leu Cys Leu Ser Leu Val Asp Arg Cys 275 280 285 Tyr Lys Ser His Ser Val Phe Ser Cys Val Leu Ala Asp Arg Tyr Cys 290 295 300 Glu Gln Gln Ile Thr Gly Val Tyr Glu Lys Ser Gly Arg Asn Pro Tyr 305 310 315 320 Asp Ile Arg Ser Lys Cys Glu Ala Glu Asp Asp Ser Gly Ala Cys Tyr 325 330 335 Gln Glu Glu Ile Tyr Ile Ser Asp Tyr Leu Asn Gln Glu Glu Val Gln 340 345 350 Arg Ala Leu Gly Thr Asp Val Ser Ser Phe Gln Gly Cys Ser Ser Asp 355 360 365 Val Gly Ile Gly Phe Ala Phe Thr Gly Asp Gly Pro Ser Pro Phe His 370 375 380 Gln Tyr Val Ala Glu Leu Leu Asp Gln Asp Ile Asn Val Leu Ile Tyr 385 390 395 400 Ala Gly Asp Lys Asp Tyr Ile Cys Asn Trp Leu Gly Asn Leu Ala Trp 405 410 415 Thr Glu Lys Leu Glu Trp Arg Tyr Asn Glu Glu Tyr Lys Lys Gln Val 420 425 430 Leu Arg Thr Trp Lys Ser Glu Glu Thr Asp Glu Thr Ile Gly Glu Thr 435 440 445 Lys Ser Tyr Gly Pro Leu Thr Tyr Leu Arg Ile Tyr Asp Ala Gly His 450 455 460 Met Val Pro His Asp Gln Pro Glu Asn Ser Leu Gln Met Val Asn Ser 465 470 475 480 Trp Ile Gln Asn Ile Ala Lys Arg Ser Arg Ile 485 490 1615 base pairs nucleic acid double linear cDNA Kluyveromyces lactis CDS 188..1417 /product= “Protease A Gene” /gene= “PRA1” 5 ATCGATAATA GAAGTGTTGA CATAACTATA TTAAAGACAG GGTAGACGGT CAGATATATA 60 GTAGTGTCAG TATTTTGAAC GGAGAGGAAC TTGATTAAAT CTATTATACA GTTTCCCCCA 120 AAATTTTTCT GAAATTGTGC CGCTAACTGT TCATTAAACG GTGCTTTCTT ACAACAAAAA 180 AATAAGC ATG CAT TTG AAT TTC CAA TCT CTT TTG CCT CTA GCT TCA TTG 229 Met His Leu Asn Phe Gln Ser Leu Leu Pro Leu Ala Ser Leu 1 5 10 TTA TTG GCT TCT TTT GAT GTT GCT GAA GCC AAG ATT CAT AAG GCC AAA 277 Leu Leu Ala Ser Phe Asp Val Ala Glu Ala Lys Ile His Lys Ala Lys 15 20 25 30 ATT CAA AAA CAT AAA TTG GAA GAC CAA TTG AAG GAT GTT CCA TTT GCC 325 Ile Gln Lys His Lys Leu Glu Asp Gln Leu Lys Asp Val Pro Phe Ala 35 40 45 GAA CAT GTG GCT CAA CTA GGT GAA AAG TAC TTA AAT AGC TTC CAA AGA 373 Glu His Val Ala Gln Leu Gly Glu Lys Tyr Leu Asn Ser Phe Gln Arg 50 55 60 GCT TAC CCT CAA GAA TCT TTC TCT AAG GAT AAC GTT GAT GTT TTC GTT 421 Ala Tyr Pro Gln Glu Ser Phe Ser Lys Asp Asn Val Asp Val Phe Val 65 70 75 GCC CCA GAA GGG TCT CAC AGT GTC CCA TTG ACC AAT TAC TTG AAT GCT 469 Ala Pro Glu Gly Ser His Ser Val Pro Leu Thr Asn Tyr Leu Asn Ala 80 85 90 CAG TAT TTC ACA GAA ATT ACT TTG GGT TCG CCA CCA CAG TCT TTT AAG 517 Gln Tyr Phe Thr Glu Ile Thr Leu Gly Ser Pro Pro Gln Ser Phe Lys 95 100 105 110 GTT ATC TTA GAC ACT GGT TCA TCA AAC TTG TGG GTT CCA AGT GCA GAA 565 Val Ile Leu Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ala Glu 115 120 125 TGT GGT TCT TTG GCA TGT TTC TTG CAC ACC AAA TAT GAC CAT GAG GCT 613 Cys Gly Ser Leu Ala Cys Phe Leu His Thr Lys Tyr Asp His Glu Ala 130 135 140 TCT AGC ACT TAC AAA GCT AAT GGT TCC GAG TTT GCT ATC CAA TAT GGT 661 Ser Ser Thr Tyr Lys Ala Asn Gly Ser Glu Phe Ala Ile Gln Tyr Gly 145 150 155 TCT GGT TCC CTT GAA GGA TAT GTG TCT CGT GAT TTG TTG ACC ATT GGG 709 Ser Gly Ser Leu Glu Gly Tyr Val Ser Arg Asp Leu Leu Thr Ile Gly 160 165 170 GAT TTA GTG ATA CCT GAC CAG GAT TTC GCT GAA GCT ACC AGC GAA CCA 757 Asp Leu Val Ile Pro Asp Gln Asp Phe Ala Glu Ala Thr Ser Glu Pro 175 180 185 190 GGT TTG GCA TTT GCC TTT GGT AAA TTC GAT GGT ATT TTG GGG TTG GCT 805 Gly Leu Ala Phe Ala Phe Gly Lys Phe Asp Gly Ile Leu Gly Leu Ala 195 200 205 TAC GAC TCC ATC TCT GTT AAC AGA ATC GTT CCA CCA GTG TAC AAC GCT 853 Tyr Asp Ser Ile Ser Val Asn Arg Ile Val Pro Pro Val Tyr Asn Ala 210 215 220 ATC AAA AAC AAA CTT TTG GAT GAC CCA GTG TTT GCC TTT TAC TTG GGT 901 Ile Lys Asn Lys Leu Leu Asp Asp Pro Val Phe Ala Phe Tyr Leu Gly 225 230 235 GAT TCT GAC AAG TCT GAA GAT GGC GGT GAA GCT TCC TTC GGT GGT ATC 949 Asp Ser Asp Lys Ser Glu Asp Gly Gly Glu Ala Ser Phe Gly Gly Ile 240 245 250 GAT GAG GAG AAG TAC ACC GGT GAA ATC ACT TGG TTG CCT GTT CGT CGT 997 Asp Glu Glu Lys Tyr Thr Gly Glu Ile Thr Trp Leu Pro Val Arg Arg 255 260 265 270 AAG GCT TAC TGG GAA GTC AAG TTT GAA GGT ATC GGT TTG GGT GAA GAA 1045 Lys Ala Tyr Trp Glu Val Lys Phe Glu Gly Ile Gly Leu Gly Glu Glu 275 280 285 TAT GCT ACT TTA GAA GGT CAT GGT GCT GCT ATC GAC ACC GGT ACC TCT 1093 Tyr Ala Thr Leu Glu Gly His Gly Ala Ala Ile Asp Thr Gly Thr Ser 290 295 300 TTG ATT GCT TTG CCA AGC GGT TTG GCT GAA ATT TTG AAC GCT GAA ATC 1141 Leu Ile Ala Leu Pro Ser Gly Leu Ala Glu Ile Leu Asn Ala Glu Ile 305 310 315 GGT GCA AAG AAG GGC TGG TCT GGT CAA TAC TCC GTT GAT TGT GAA TCT 1189 Gly Ala Lys Lys Gly Trp Ser Gly Gln Tyr Ser Val Asp Cys Glu Ser 320 325 330 AGA GAT AGT CTA CCA GAC TTA ACT TTG AAT TTC AAC GGT TAC AAC TTC 1237 Arg Asp Ser Leu Pro Asp Leu Thr Leu Asn Phe Asn Gly Tyr Asn Phe 335 340 345 350 ACT ATT ACC GCA TAC GAT TAC ACT TTG GAA GTC TCT GGG TCT TGT ATC 1285 Thr Ile Thr Ala Tyr Asp Tyr Thr Leu Glu Val Ser Gly Ser Cys Ile 355 360 365 TCT GCA TTC ACT CCA ATG GAC TTC CCA GAA CCA GTT GGT CCC TTG GCC 1333 Ser Ala Phe Thr Pro Met Asp Phe Pro Glu Pro Val Gly Pro Leu Ala 370 375 380 ATT ATT GGT GAT GCC TTC CTA CGT AAA TAC TAC TCC ATT TAT GAT ATT 1381 Ile Ile Gly Asp Ala Phe Leu Arg Lys Tyr Tyr Ser Ile Tyr Asp Ile 385 390 395 GGT CAT GAT GCA GTT GGT TTG GCC AAG GCT GCC TAATTGTTAA AAAAGCGATC 1434 Gly His Asp Ala Val Gly Leu Ala Lys Ala Ala 400 405 410 GAATTGTAAC CTTTTGAATT GGAGTTCAGC TTCTATTAAC TCGACAACTC TAAAAAAATA 1494 ATTAAATAAG ACGGTTAACT TACTGCTATA TTAATTGAAT GTCAGTTTCA CAAATCGAAT 1554 TAGCTAACAA AGTATAACAA CACTTGGTGA CAAATAAACC TTAAAATACC TGGCAGAATT 1614 C 1615 409 amino acids amino acid linear protein not provided 6 Met His Leu Asn Phe Gln Ser Leu Leu Pro Leu Ala Ser Leu Leu Leu 1 5 10 15 Ala Ser Phe Asp Val Ala Glu Ala Lys Ile His Lys Ala Lys Ile Gln 20 25 30 Lys His Lys Leu Glu Asp Gln Leu Lys Asp Val Pro Phe Ala Glu His 35 40 45 Val Ala Gln Leu Gly Glu Lys Tyr Leu Asn Ser Phe Gln Arg Ala Tyr 50 55 60 Pro Gln Glu Ser Phe Ser Lys Asp Asn Val Asp Val Phe Val Ala Pro 65 70 75 80 Glu Gly Ser His Ser Val Pro Leu Thr Asn Tyr Leu Asn Ala Gln Tyr 85 90 95 Phe Thr Glu Ile Thr Leu Gly Ser Pro Pro Gln Ser Phe Lys Val Ile 100 105 110 Leu Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ala Glu Cys Gly 115 120 125 Ser Leu Ala Cys Phe Leu His Thr Lys Tyr Asp His Glu Ala Ser Ser 130 135 140 Thr Tyr Lys Ala Asn Gly Ser Glu Phe Ala Ile Gln Tyr Gly Ser Gly 145 150 155 160 Ser Leu Glu Gly Tyr Val Ser Arg Asp Leu Leu Thr Ile Gly Asp Leu 165 170 175 Val Ile Pro Asp Gln Asp Phe Ala Glu Ala Thr Ser Glu Pro Gly Leu 180 185 190 Ala Phe Ala Phe Gly Lys Phe Asp Gly Ile Leu Gly Leu Ala Tyr Asp 195 200 205 Ser Ile Ser Val Asn Arg Ile Val Pro Pro Val Tyr Asn Ala Ile Lys 210 215 220 Asn Lys Leu Leu Asp Asp Pro Val Phe Ala Phe Tyr Leu Gly Asp Ser 225 230 235 240 Asp Lys Ser Glu Asp Gly Gly Glu Ala Ser Phe Gly Gly Ile Asp Glu 245 250 255 Glu Lys Tyr Thr Gly Glu Ile Thr Trp Leu Pro Val Arg Arg Lys Ala 260 265 270 Tyr Trp Glu Val Lys Phe Glu Gly Ile Gly Leu Gly Glu Glu Tyr Ala 275 280 285 Thr Leu Glu Gly His Gly Ala Ala Ile Asp Thr Gly Thr Ser Leu Ile 290 295 300 Ala Leu Pro Ser Gly Leu Ala Glu Ile Leu Asn Ala Glu Ile Gly Ala 305 310 315 320 Lys Lys Gly Trp Ser Gly Gln Tyr Ser Val Asp Cys Glu Ser Arg Asp 325 330 335 Ser Leu Pro Asp Leu Thr Leu Asn Phe Asn Gly Tyr Asn Phe Thr Ile 340 345 350 Thr Ala Tyr Asp Tyr Thr Leu Glu Val Ser Gly Ser Cys Ile Ser Ala 355 360 365 Phe Thr Pro Met Asp Phe Pro Glu Pro Val Gly Pro Leu Ala Ile Ile 370 375 380 Gly Asp Ala Phe Leu Arg Lys Tyr Tyr Ser Ile Tyr Asp Ile Gly His 385 390 395 400 Asp Ala Val Gly Leu Ala Lys Ala Ala 405 224 amino acids amino acid linear peptide internal Kluyveromyces lactis 7 Arg Ser Asn Gly Ser Gly Thr Met Ser Asp Val Val Lys Gly Val Glu 1 5 10 15 Tyr Val Ala Glu Ala His Lys Lys Ala Val Glu Glu Gln Lys Lys Gly 20 25 30 Phe Lys Gly Ser Thr Ala Asn Met Ser Leu Gly Gly Gly Lys Ser Pro 35 40 45 Ala Leu Asp Leu Ala Val Asn Ala Ala Val Lys Ala Gly Val His Phe 50 55 60 Ala Val Ala Ala Gly Asn Glu Asn Gln Asp Ala Cys Asn Thr Ser Pro 65 70 75 80 Ala Ala Ala Glu Asn Ala Ile Thr Val Gly Ala Ser Thr Leu Ser Asp 85 90 95 Glu Arg Ala Tyr Phe Ser Asn Trp Gly Lys Cys Val Asp Ile Phe Gly 100 105 110 Pro Gly Leu Asn Ile Leu Ser Thr Tyr Ile Gly Ser Asp Thr Ala Thr 115 120 125 Ala Thr Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Val Gly Leu 130 135 140 Leu Thr Tyr Phe Leu Ser Leu Gln Pro Asp Ala Asp Ser Glu Tyr Phe 145 150 155 160 His Ala Ala Gly Gly Ile Thr Pro Ser Gln Leu Lys Lys Lys Leu Ile 165 170 175 Asp Phe Ser Thr Lys Asn Val Leu Ser Asp Leu Pro Glu Asp Thr Val 180 185 190 Asn Tyr Leu Ile Tyr Asn Gly Gly Gly Gln Asp Leu Asp Asp Leu Trp 195 200 205 Gly Lys Asp Tyr Ser Ile Gly Lys Glu Pro Ser Ala Asn Pro Glu Phe 210 215 220 225 amino acids amino acid linear peptide internal Saccharomyces cerevisiae 8 Arg Ser Asn Gly Ser Gly Thr Met Ser Asp Val Val Lys Gly Val Glu 1 5 10 15 Tyr Ala Ala Lys Ala His Gln Lys Glu Ala Gln Glu Lys Lys Lys Gly 20 25 30 Phe Lys Gly Ser Thr Ala Asn Met Ser Leu Gly Gly Gly Lys Ser Pro 35 40 45 Ala Leu Asp Leu Ala Val Asn Ala Ala Val Glu Val Gly Ile His Phe 50 55 60 Ala Val Ala Ala Gly Asn Glu Asn Gln Asp Ala Cys Asn Thr Ser Pro 65 70 75 80 Ala Ser Ala Glu Lys Ala Ile Thr Val Gly Ala Ser Thr Leu Ser Asp 85 90 95 Asp Arg Ala Tyr Phe Ser Asn Trp Gly Lys Cys Val Asp Val Phe Ala 100 105 110 Pro Gly Leu Asn Ile Leu Ser Thr Tyr Ile Gly Ser Asp Asp Ala Thr 115 120 125 Ala Thr Leu Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Leu 130 135 140 Leu Thr Tyr Phe Leu Ser Leu Gln Pro Gly Ser Asp Ser Glu Phe Phe 145 150 155 160 Glu Leu Gly Glu Asp Ser Leu Thr Pro Gln Gln Leu Lys Lys Lys Leu 165 170 175 Ile His Tyr Ser Thr Lys Asp Ile Leu Phe Asp Ile Pro Glu Asp Thr 180 185 190 Pro Asn Val Leu Ile Tyr Asn Gly Gly Gly Gln Asp Leu Ser Ala Phe 195 200 205 Trp Asn Lys Asp Thr Lys Lys Ser His Ser Ser Gly Phe Lys Gln Glu 210 215 220 Leu 225 17 base pairs nucleic acid single linear other nucleic acid not provided 9 TGACACTCAA AATAGCG 17 17 base pairs nucleic acid single linear other nucleic acid not provided 10 AATATCTCTC ACTTGAT 17 21 base pairs nucleic acid single linear other nucleic acid not provided 11 GACCTATGGG GTAAGGATTA C 21 18 base pairs nucleic acid single linear other nucleic acid not provided 12 GCTTCGGCAA CATATTCG 18 20 base pairs nucleic acid single linear other nucleic acid not provided 13 CTTCTTGGAG TTGTTCTTCG 20 30 base pairs nucleic acid single linear other nucleic acid not provided 14 TGGCAAGACA TCCGTCCACG CCTTATTACC 30 18 base pairs nucleic acid single linear other nucleic acid not provided 15 CTGTTGATAA GGTGGTCC 18 20 base pairs nucleic acid single linear other nucleic acid not provided 16 CAAGCGTGTA ATCGTATGGC 20 30 base pairs nucleic acid single linear other nucleic acid not provided 17 GAAATGCATA AGCTCTTGCC ATTCTCACCG 30 

We claim:
 1. A Kluyverornyces Kluyveromyces yeast comprising a genetic modification in its PRA1, PRB1 or PRC1 gini gene, wherein said modification results in loss of protease activity encoded by said gene.
 2. The Kluyverormyces yeast according to claim 1, wherein the modification is in a) protein coding region of said gene; b) a region responsible for transcriptional regulation of said gene; or c) both a protein coding region of said gene and a region responsible for transcriptional regulation of said gene.
 3. The Kluyveromyces yeast according to claim 1, selected from the group consisting of K. lactis, K. fragilis, K, drosophilarum, and K. waltii.
 4. A process for preparing the Kluyveromyces yeast of claim 1 comprising replacing all or part of said gene with a modified version thereof.
 5. A Kluyveromyces yeast comprising a genetic modification in its PRA1, PRB1, or PRC1 gene, wherein said modification results in loss of protease activity encoded by said gene, and an exogenous DNA sequence comprising a nucleic acid sequence encoding at least one protein.
 6. The Kluyveromyces yeast according to claim 5, wherein the exogenous DNA sequence further comprises a region for initiation of transcription and translation operably linked to the nucleic acid sequence encoding the at least one protein.
 7. The Kluyveromyces yeast according to claim 5, wherein the exogenous sequence comprises a region encoding an exporting sequence for secretion of the protein.
 8. The Kluyveromyces yeast according to claim 5, wherein the exogenous DNA sequence is part of an autonomously replicating vector or is integrated into a chromosome.
 9. A process for producing recombinant proteins comprising culturing the Kluyverornyces Kluyveromyces yeast of claim 5 under conditions for expression of said exogenous DNA sequence.
 10. A process for producing recombinant proteins comprising culturing the Kluyveromyces yeast of claim 6 under conditions for expression of said exogenous DNA sequence.
 11. A process for producing recombinant proteins comprising culturing the Kluyverormyces yeast of claim 7 under conditions for expression of said exogenous DNA sequence.
 12. The Kluyveromyces yeast according to claim 5, wherein the exogenous DNA sequence encodes a protein of pharmaceutical interest.
 13. A process for producing recombinant proteins comprising culturing the Kluyveromyces yeast of claim 12 under conditions for expression of said exogenous DNA sequence. 